Systems and methods for modulating chromosomal rearrangements

Abstract

The present disclosure provides systems and methods for modulating the occurrence of chromosomal rearrangements, e.g., translocations, in a cell during genome editing. Embodiments are provided for reducing the occurrence of unwanted chromosomal rearrangements, and for increasing the occurrence of desired chromosomal rearrangements.

The invention claimed is: 
     
       1. A method of altering a cell at two target nucleic acids in the cell, the method comprising the step of delivering to the cell two ribonucleoprotein (RNP) complexes, wherein a first RNP complex comprises a SpCas9 RNA-guided nuclease, and wherein a second RNP complex comprises an Acidaminococcus sp. Cpf1 nuclease, thereby altering the cell at the two target nucleic acids. 
     
     
       2. A method of reducing the risk of translocations in a cell when the cell is altered at two target nucleic acids, the method comprising delivering to the cell two RNP complexes, wherein a first RNP complex comprises a SpCas9 RNA-guided nuclease, and wherein a second RNP complex comprises an Acidaminococcus sp. Cpf1 nuclease, thereby reducing the risk of translocations in the cell. 
     
     
       3. The method of  claim 2 , wherein the translocation may occur between an on-target site and an off-target site. 
     
     
       4. The method of  claim 1 , wherein the two RNP complexes are delivered to the cell sequentially in any order, or simultaneously. 
     
     
       5. A method of altering a cell at a first target nucleic acid and a second target nucleic acid, comprising the steps of:
 forming at least one single- or double-stranded break at a first cleavage site in the first target nucleic acid by delivering to the cell a ribonucleoprotein (RNP) complex comprising a first RNA-guided nuclease and a first guide RNA (gRNA) capable of directing the first RNA-guided nuclease to the first target nucleic acid, wherein the first cleavage site is repaired by at least one DNA repair pathway to produce an altered first target nucleic acid; and 
 forming at least one single- or double-stranded break at a second cleavage site in the second target nucleic acid by delivering to the cell a second RNA-guided nuclease expressed in the cell from an exogenous nucleic acid encoding the second RNA-guided nuclease, wherein the second cleavage site is repaired by at least one DNA repair pathway to produce an altered second target nucleic acid, 
 wherein the first RNA-guided nuclease is SpCas9 nuclease and the second RNA-guided nuclease is an  Acidaminococcus  sp. Cpf1 nuclease, and wherein the first and the second RNA complexes may be delivered simultaneously or sequentially in any order. 
 
     
     
       6. A method of altering a cell at a first target nucleic acid and a second target nucleic acid, comprising the steps of:
 forming at least one single- or double-stranded break at a first cleavage site in the first target nucleic acid by delivering to the cell a first ribonucleoprotein (RNP) complex comprising a first RNA-guided nuclease and a first guide RNA (gRNA) capable of directing the first RNA-guided nuclease to the first cleavage site in the first target nucleic acid, wherein the first RNA-guided nuclease is an SpCas9 nuclease, and wherein the first cleavage site is repaired by at least one DNA repair pathway to produce an altered first target nucleic acid; and 
 after a period of time sufficient for repair of the first cleavage site, forming at least one single- or double-stranded break at a second cleavage site by delivering to the cell a second ribonucleoprotein (RNP) complex comprising a second RNA-guided nuclease and a second guide RNA (gRNA) capable of directing the second RNA-guided nuclease to the second cleavage site in the second target nucleic acid, wherein the second RNA-guided nuclease is an  Acidaminococcus  sp. Cpf1 nuclease, and wherein the second cleavage site is repaired by at least one DNA repair pathway to produce an altered second target nucleic acid, thereby altering the cell. 
 
     
     
       7. The method of  claim 6 , wherein the first RNP complex and the second RNP complex are delivered in different amounts. 
     
     
       8. The method of  claim 7 , wherein the concentration of the second RNP complex is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold or 50-fold lower than the amount of the first RNP complex. 
     
     
       9. The method of  claim 6 , wherein the time sufficient for repair of the first cleavage site is at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours. 
     
     
       10. The method of  claim 5 , wherein the cell is a T cell, an NK cell, an embryonic stem cell, an induced pluripotent stem cell (iPSC), a CD34+ cell, or a hematopoietic stem/progenitor cell (HSPC). 
     
     
       11. The method of  claim 10 , wherein the cell is a T cell, and the first target nucleic acid is selected from the group consisting of TRAC, TRBC, CIITA, and B2M. 
     
     
       12. The method of  claim 11 , wherein the second target nucleic acid is different from the first target nucleic acid. 
     
     
       13. The method of  claim 12 , wherein the second target nucleic acid is selected from the group consisting of TRAC, TRBC, CIITA, and B2M. 
     
     
       14. The method of  claim 1 , wherein the method is performed ex vivo. 
     
     
       15. The method of  claim 1 , wherein the method is performed in vivo. 
     
     
       16. The method of  claim 1 , wherein the cell is a T cell, an NK cell, an embryonic stem cell, an induced pluripotent stem cell (iPSC), a CD34+ cell, or a hematopoietic stem/progenitor cell (HSPC). 
     
     
       17. The method of  claim 1 , further comprising contacting the cell with an exogenous oligonucleotide. 
     
     
       18. The method of  claim 17 , wherein the exogenous oligonucleotide is an exogenous oligonucleotide donor template.